Method and apparatus for chemical and biochemical reactions using photo-generated reagents

ABSTRACT

This invention provides method and apparatus for performing chemical and biochemical reactions in solution using in situ generated photo-products as reagent or co-reagent. Specifically, the method and apparatus of the present invention have applications in parallel synthesis of molecular sequence arrays on solid surfaces.

This application claims the benefit of U.S. Provisional Application No.60/074,368, filed on Feb. 11, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of chemical and biochemicalreactions. More specifically, the present invention relates to parallelsynthesis and assay of a plurality of organic and bio-organic moleculeson a substrate surface in accordance with a predetermined spatialdistribution pattern. Methods and apparatus of the present invention areuseful for preparing and assaying very-large-scale arrays of DNA and RNAoligonucleotides, peptides, oligosacchrides, phospholipids and otherbiopolymers and biological samples on a substrate surface.

2. Description of the Related Art

Development of modern medicine, agriculture, and materials imposesenormous demands on technological and methodological progress toaccelerate sample screening in chemical and biological analysis.Development of parallel processes on a micro-scale is critical to theprogress. Many advances have been made in this area using parallelsynthesis, robotic spotting, inkjet printing, and microfluidics(Marshall et al., Nature Biotech. 16, 27-31 (1998)). Continued effortsare sought for more reliable, flexible, faster, and inexpensivetechnologies.

For high-throughput screening applications, a promising approach is theuse of molecular microarray (MMA) chips, specifically biochipscontaining high-density arrays of biopolymers immobilized on solidsurfaces. These biochips are becoming powerful tools for exploringmolecular genetic and sequence information (Marshall et al., NatureBiotech. 16, 27-31 (1998) and Ramsay, Nature Biotech. 16, 40-44 (1998)).Target molecules have been hybridized to DNA oligonucleotides and cDNAprobes on biochips for determining nucleotide sequences, probingmultiplex interactions of nucleic acids, identifying gene mutations,monitoring gene expression, and detecting pathogens. Schena, et al.,Science 270, 467-460 (1995); Lockhart et al., Nature Biotech. 14,1675-1680; Weiler, Nucleic Acids Res. 25, 2792-2799 (1997); de Saizieuet al., Nature Biotech. 16, 45-48; Drmanc et al., Nature Biotech. 16,54-58. The continued development of biochip technology will have asignificant impact on the fields of biology, medicine, and clinicaldiagnosis.

Prior art biochip-fabrication includes direct on-chip synthesis (makingseveral sequences at a time) using inkjets, direct on-chip parallelsynthesis (making the whole array of sequences simultaneously) usingphotolithography, and immobilization of a library of pre-synthesizedmolecules using robotic spotting (Ramsay, Nature Biotech. 16, 40-44(1998)). Light-directed on-chip parallel synthesis has been used in thefabrication of very-large-scale oligonucleotide arrays with up to onemillion sequences on a single chip.

Two major methods have been disclosed: synthesis using photolabile-groupprotected monomers (Pirrung et al., U.S. Pat. No. 5,143,854 (1992);Fodor et al., U.S. Pat. No. 5,424,186 (1995)) and synthesis usingchemical amplification chemistry (Beecher et al., PCT Publication No. WO98/20967 (1997)). Both methods involve repetitive steps of deprotection,monomer coupling, oxidation, and capping. Photomasks are used to achieveselective light exposure in predetermined areas of a solid substratesurface, on which oligonucleotide arrays are synthesized.

For the synthesis process involving photolabile-protecting groups, thephotolabile-protecting groups are cleaved from growing oligonucleotidemolecules in illuminated surface areas while in non-illuminated surfaceareas the protecting groups on oligonucleotide molecules are notaffected. The substrate surface is subsequently contacted with asolution containing monomers having a unprotected first reactive centerand a second reactive center protected by a photolabile-protectinggroup. In the illuminated surface areas, monomers couple via theunprotected first reactive center with the deprotected oligonucleotidemolecules. However, in the non-illuminated surface areasoligonucleotides remain protected with the photolabile-protecting groupsand, therefore, no coupling reaction takes place. The resultingoligonucleotide molecules after the coupling are protected byphotolabile protecting groups on the second reactive center of themonomer. Therefore, one can continue the above photo-activated chainpropagation reaction until all desired oligonucleotides are synthesized.

For the synthesis process involving chemical amplification chemistry, aplaner substrate surface is linked with oligonucleotide molecules(through appropriate linkers) and is coated with a thin (a fewmicrometers) polymer or photoresist layer on top of the oligonucleotidemolecules. The free end of each oligonucleotide molecule is protectedwith an acid labile group. The polymer/photoresist layer contains aphoto-acid precursor and an ester (an enhancer), which, in the presenceof H⁺, dissociates and forms an acid. During a synthesis process, acidsare produced in illuminated surface areas within the polymer/photoresistlayer and acid-labile protecting groups on the ends of theoligonucleotide molecules are cleaved. The polymer/photoresist layer isthen stripped using a solvent or a stripping solution to expose theoligonucleotide molecules below. The substrate surface is then contactedwith a solution containing monomers having a reactive center protectedby an acid-labile protecting group. The monomers couple via theunprotected first reactive center only with the deprotectedoligonucleotide molecules in the illuminated areas. In thenon-illuminated areas, oligonucleotide molecules still have theirprotection groups on and, therefore, do not participate in couplingreaction. The substrate is then coated with a photo-acid-precursorcontaining polymer/photoresist again. The illumination, deprotection,coupling, and polymer/photoresist coating steps are repeated untildesired oligonucleotides are obtained.

There are significant drawbacks in the method involvingphotolabile-protecting groups: (a) the chemistry used isnon-conventional and the entire process is extremely complicated; and(b) the technique suffer from low sequence fidelity due to chemistrycomplications.

The method of using chemical amplification chemistry has its limitationsas well: (a) The method requires application of a polymer/photoresistlayer and is not suitable for reactions performed in solutions routinelyused in chemical and biochemical reactions since there is no measureprovided for separating sites of reaction on a solid surface. (b) Incertain circumstances, destructive chemical conditions required for pre-and post-heating and stripping the polymer/photoresist layer cause thedecomposition of oligonucleotides on solid surfaces. (c) The entireprocess is labor intensive and difficult to automate due to therequirement for many cycles (up to 80 cycles if 20-mers aresynthesized!) of photoresist coating, heating, alignment, light exposureand stripping. (d) The method is not applicable to a broad range ofbiochemical reactions or biological samples to which a photo-generatedreagent is applied since embedding of biological samples in apolymer/photoresist layer may be prohibitive.

Additional limitations are linked to the use of photomasks in the abovetwo methods: (a) Setup for making a new chip is very expensive due to alarge number of photomasks that have to be made. (b) Photolithographyequipment is expensive and, therefore, can not be accessed by manyinterested users. (c) Photolithography processes have to be conducted inan expensive cleanroom facility and require trained technical personnel.(d) The entire process is complicated and difficult to automate. Theselimitations undermine the applications of oligonucleotide chips and thedevelopment of the various MMA-chips.

Therefore, there is a genuine need for the development of chemicalmethods and synthesis apparatus that are simple, versatile,cost-effective, easy to operate, and that can afford molecular arrays ofimproved purity.

SUMMARY OF THE INVENTION

The present invention provides methods and apparatus for performingchemical and biochemical reactions in solution using in situ generatedphoto-products as reagents or co-reagents. These reactions arecontrolled by irradiation, such as with UV or visible light. Unlessotherwise indicated, all reactions described herein occur in solutionsof at least one common solvent or a mixture of more than one solvent.The solvent can be any conventional solvent traditionally employed inthe chemical reaction, including but not limited to such solvents asCH₂Cl₂, CH₃CN, toluene, hexane, CH₃OH, H₂O, and/or an aqueous solutioncontaining at least one added solute, such as NaCl, MgCl₂, phosphatesalts, etc. The solution is contained within defined areas on a solidsurface containing an array of reaction sites. Upon applying a solutioncontaining at least one photo-generated reagent (PGR) precursor(compounds that form at least one intermediate or product uponirradiation) on the solid surface, followed by projecting a lightpattern through a digital display projector onto the solid surface, PGRforms at illuminated sites; no reaction occurs at dark (i.e.,non-illuminated) sites. PGR modifies reaction conditions and may undergofurther reactions in its confined area as desired. Therefore, in thepresence of at least one photo-generated reagent (PGR), at least onestep of a multi-step reaction at a specific site on the solid surfacemay be controlled by radiation, such as light, irradiation. Hence, thepresent invention has great potential in the applications of parallelreactions, wherein at each step of the reaction only selected sites in amatrix or array of sites are allowed to react.

The present invention also provides an apparatus for performing thelight controlled reactions described above. One of the applications ofthe instrument is to control reactions on a solid surface containing aplurality of isolated reaction sites, such as wells (the reactor). Lightpatterns for effecting the reactions are generated using a computer anda digital optical projector (the optical module). Patterned light isprojected onto specific sites on the reactor, where light controlledreactions occur.

One of the applications of the present invention provides in situgeneration of chemical/biochemical reagents that are used in thesubsequent chemical and biochemical reactions in certain selected sitesamong the many possible sites present. One aspect of the invention is tochange solution pH by photo-generation of acids or bases in a controlledfashion. The pH conditions of selected samples can be controlled by theamount of photo-generated acids or bases present. The changes in pHconditions effect chemical or biochemical reactions, such as byactivating enzymes and inducing couplings and cross-linking throughcovalent or non-covalent bond formation between ligand molecules andtheir corresponding receptors.

In other aspects of the present invention, photo-generated reagentsthemselves act as binding molecules that can interact with othermolecules in solution. The concentration of the binding molecules isdetermined by the dose of light irradiation and, thus, the ligandbinding affinity and specificity in more than one system can be examinedin parallel. Therefore, the method and apparatus of the presentinvention permits investigating and/or monitoring multiple processsessimultaneously and high-throughput screening of chemical, biochemical,and biological samples.

Another important aspect of the present invention parallel synthesis ofbiopolymers, such as oligonucleotides and peptides, wherein the methodand instrument of the present invention are used for selectivedeprotection or coupling reactions. These reactions permit controlledfabrication of diverse biopolymers on solid surfaces. These molecularmicroarray chips (MMA-chips) are used in a wide range of fields, such asfunctional genomics, diagnosis, therapeutics and genetic agriculture andfor detecting and analyzing gene sequences and their interactions withother molecules, such as antibiotics, antitumor agents, oligosacchrides,and proteins. These and other aspects demonstrate features andadvantages of the present invention. Further details are made clear byreference to the remaining portions of the specification and theattached drawings.

The method of the present invention represents fundamental improvementscompared to the method of prior arts for parallel synthesis of DNAoligonucleotide arrays (Pirrung et al., U.S. Pat. No. 5,143,854 (1992);Fodor et al., U.S. Pat. No. 5,424,186 (1995); Beecher et al., PCTPublication No. WO 98/20967 (1997)). The present inventionadvantageously employs existing chemistry, replacing at least one of thereagents in a reaction with a photo-reagent precursor. Therefore, unlikemethods of the prior art, which require monomers containing photolabileprotecting groups or a polymeric coating layer as the reaction medium,the present method uses monomers of conventional chemical and requiresminimal variation of the conventional synthetic chemistry and protocols.

The improvements made possible by the present invention have significantconsequences: (a) The synthesis of sequence arrays using the method ofthe present invention can be easily integrated into an automated DNA/RNAsynthesizer, so that the process of the present invention is muchsimpler and costs much less. (b) Conventional chemistry adopted by thepresent invention routinely achieves better than 98% yield per stepsynthesis of oligonucleotides, which is far better than the 85-95% yieldobtained by the previous method of using photolabile protecting groups.Pirrung et al., J. Org. Chem. 60, 6270-6276, (1995); McGall et al., J.Am. Chem. Soc. 119, 5081-5090 (1997); McGall et al., Proc. Natl. Acad.Sci. USA 93, 13555-13560 (1996). The improved stepwise yield is criticalfor synthesizing high-quality oligonucleotide arrays for diagnostic andclinical applications. (c) Yield of photo-generated products (PGR) isnot a major concern in the method of the present invention in contrastto that of the prior art method on incomplete deprotection onphotolabile protecting groups. (d) The synthesis process of the presentinvention can be monitored using conventional chemistry for qualitycontrol; this is not possible using the methods of the prior art. (e)The method of the present invention is easily expandable to thesynthesis of other types of molecular microarrays, such asoligonucleotides containing modified residues, 3′-oligonucleotides (asopposed to 5′-oligonucleotides obtained in a normal synthesis),peptides, oligosacchrides, combinatory organic molecules, and the like.These undertakings would be an insurmountable task using prior arttechniques requiring monomers containing photolabile-protecting groups.The prior art methods require development of new synthetic proceduresfor each monomer type. In the present invention, modified residues andvarious monomers that are commercially available can be employed. (f)The present invention can be applied to all types of reactions and isnot limited to polymeric reaction media as is the prior art method usingchemical amplification reactions. (g) Additionally, the reaction timefor each step of synthesis using the conventional oligonucleotidechemistry (5 min. per step) is much shorter than methods usingphotolabile blocked monomers (>15 min. per step).

Optical patterning in prior art biochip fabrication uses standardphotomask-based lithography tools, Karl et al., U.S. Pat. No. 5,593,839(1997). In general, the number and pattern complexity of the masksincrease as the length and variety of oligomers increase. For example,4×12=48 masks are required to synthesize a subset of dodecanucleotides,and this number may be larger depending on the choice of custom chip. Tomake a new set of sequences, a new set of masks have to be prepared.More critical is the high precision alignment (on the order of <10 μmresolution) of the successive photomasks, a task that is impossible toachieve without specialized equipment and technical expertise. Thetechnology is only semi-automatic and the method is clearly inflexibleand expensive. In addition, the photomask-fabrication process requiresexpensive cleanroom facilities and demands special technical expertisein microelectronic fields. Therefore, the entire chip-fabricationprocess is inaccessible to most in the research community.

The present invention replaces the photomasks with a computer-controlledspatial optical modulator so that light patterns for photolithographycan be generated by a computer in the same way as it displaysblack-and-white images on a computer screen. This modification providesmaximum flexibility for synthesizing any desirable sequence array andsimplifies the fabrication process by eliminating the need forperforming mask alignment as in the conventional photolithography, whichis time consuming and prone to alignment errors. In addition, both theoptical system and the reactor system of the present invention arecompact and can be integrated into one desktop enclosure. Such aninstrument can be fully controlled by a personal computer so that anybench chemists can make biochips of their own sequence design in a waythat is similar to bio-oligomer synthesis using a synthesizer. Moreover,the instrument can be operated in any standard chemical lab without theneed for a cleanroom. The present invention can also be easily adoptedto streamline production of large quantities of standard biochips or afixed number of specialized biochips by automated production lines.Obviously, the cost of making biochips can be significantly reduced bythe method and apparatus of the present invention and, therefore, theaccessibility of the biochip technology to research and biomedicalcommunities can be significantly increased.

Most importantly, the method of the present invention usingphoto-generated reagents in combination with a computer-controlledspatial optical modulator makes MMA-chip fabrication a routine process,overcoming limitations of the prior art methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of oligonucleotide synthesis using photo-generatedacids. L—linker group; P_(a)—acid-labile protecting group;H⁺—photo-generated acid; T, A, C, and G—nucleotide phophoramiditemonomers; hν—light exposure. In this Figure, SEQ ID NOS:2-5 are shown.

FIG. 2 is a drawing of the deprotection process using photo-generatedacids in oligonucleotide synthesis.

FIG. 3 is a drawing of oligonucleotide synthesis using photo-generatedreagents. The process is the same as shown in FIG. 1 except that aphoto-generated activator, such as dimethoxybenzoinyltetrazole, is used,while the deprotection step is accomplished using a conventional acid.In this Figure, SEQ ID NOS:2-5 are shown.

FIG. 4 is a drawing of amino acid deprotection using photo-generatedacids or photo-generated bases. Boc=butyloxylcarbonyl;Fmoc=fluoroenylmethyloxycarbonyl.

FIG. 5 is a drawing of peptide synthesis using photo-generated acids.L—linker group; P_(a)—acid-labile protecting group; F, Q, D, Y, S, andA—representative Boc-protected amino acids; hν—light exposure. In thisFigure, SEQ ID NOS:6-9 are shown.

FIG. 6 is a drawing of peptide synthesis using photo-generated bases.L—linker group; P_(a)—acid-labile protecting group; F, Q, D, Y, S, andA—representative Fmoc-protected amino acids; hν—light exposure. In thisFigure, SEQ ID NOS:6-9 are shown.

FIG. 7 is a drawing of carbohydrate synthesis using both photo-generatedacids and photo-generated bases at various of reaction steps.

FIG. 8A is a schematic illustration of the synthesis apparatus using amicromirror array modulator.

FIG. 8B is a schematic illustration of the synthesis apparatus using areflective LCD array modulator.

FIG. 8C is a schematic illustration of the synthesis apparatus using atransmissive LCD array modulator.

FIG. 9A illustrates an isolation mechanism using microwell structures ona back cover.

FIG. 9B illustrates an isolation mechanism using microwell structures ona substrate.

FIG. 9C illustrates an isolation mechanism using a patterned non-wettingfilm on a substrate.

FIG. 10 is an exploded schematic of a reactor cartridge and an enlargedview of reaction-wells.

FIG. 11A is a schematic illustration of the deprotection reaction in apartially masked reaction-well.

FIG. 11B is a schematic illustration of the deprotection reaction with areaction-well being partially exposed.

FIG. 12 illustrates a stepping mechanism for parallel synthesis of aplurality of arrays.

FIGS. 13A and 13B are linear and log scale plots of H₃O⁺ chemical shift(ppm) versus light irradiation time (min) measured from a samplecontaining a photo-acid precursor, respectively.

FIGS. 14A1 and 14A2 show the HPLC profiles of non-irradiated andirradiated DNA nucleosides in the presence of a photo-generated acidprecursor, respectively.

FIGS. 14B1 and 14B2 show the HPLC profiles of non-irradiated andirradiated RNA nucleosides in the presence of a photo-generated acidprecursor, respectively.

FIGS. 15A1 and 15A2 show the HPLC profiles of the DNA oligomer TTTTsynthesized using a conventional acid and a photo-generated acid (PGA),respectively.

FIGS. 15B1 and 15B2 show the HPLC profiles of the DNA oligomer TTTTTTTTand ACGTACGT synthesized using a PGA, respectively.

FIGS. 16A and 16B show the HPLC profiles of amino acids (Tyr) obtainedfrom the deprotection of t-Boc-Tyr using conventional trifluoroaceticacid (TFA) and photogenerated acid (PGA), respectively.

FIG. 17A illustrates a fabrication process for making microwells on aflat substrate.

FIG. 17B is an enlarged photograph of microwells on a glass substrate.

FIG. 18A illustrates a fabrication process for making a non-wetting-filmpattern on a flat substrate.

FIG. 18B is an enlarged photograph of methanol-droplets formed on aglass surface containing non-wetting film patterns.

FIG. 19 is a fluorescence image of fluorescein tagged thymine grown on anon-wetting film patterned glass plate.

DETAILED DESCRIPTION OF THE INVENTION Method for Chemical/BiochemicalReactions Using Photo-Generated Reagents (PGR)

The present invention provides a method for solution based photochemicalreactions involving reagents generated in situ by irradiation. Aconventional chemical/biochemical reaction occurs between at least onereactant (generically denoted as “A”) and at least one reagent(generically denoted as “R”) to give at least one product as depictedbelow:

A+R→A′+R′

The present invention is to provide reaction conditions that arecontrolled by irradiation with light. Mainly, the R in the reactionabove is photo-generated. The photo-generated reagent (PGR) functionsthe same as a reagent conventionally used and, thus, the reactionproceeds in an otherwise conventional way. The overall photo-controlledreaction is depicted below.

In some embodiments of the present invention, PGR precursors (Table 1)are photo-generated acid precursors that yield H⁺ in the form of R₁CO₂H,R₁PO₃H, R₁SO₃H, H⁺X⁻ (R₁=H, alkyl (C₁-C₁₂), aryl (aromatic structurescontaining phenyl), or their substituted derivatives(substitutions=halogen atoms, NO₂, CN, OH, CF₃, C(O)H, C(O)CH₃, C(O)R₂,SO₂CH₃, SO₂R₂, OCH₃, OR₂, NH₂, NHR₂, NR₂R₃ (R₂ and R₃=alkyl or aryl(C₁-C₁₂)); X=halogen atoms, inorganic salt ions) or the like.Photo-generated acids are also complexes, such as M_(m)X_(n) (Lewisacids, m and n are number of atoms) formed upon irradiation. In otherembodiments of the present invention, PGR precursors (Table 1) arephoto-generated base precursors that yield a base, such as an amine, anoxide or the like, upon irradiation.

TABLE 1A Examples of Photo-Generated Reagent Precursors and TheirProducts Photo-Reagent Reagent Precursor Chemical Structure Generateddiazonium salts

B(R₁)₃, Al(R₁)₃ X = B(R₁)₄, Al(R₁)₄ (R₁ = halogen); R = H, halogen, NO₂,CN, SO₂R₅, OH, OCH₃, SCH₃, CF₃, OR₅, SR₅, CH₃, t-butyl, C₁-C₁₂- alkyl,aryl and their substituted derivatives^(a), NH₂, HNR₅, N(R₅)₂, (R₅ =C₁-C₁₂-alkyl, aryl and their substituted derivatives^(a)); COR₆ (R₆ = H,NH₂, HNR₅, OR₅, C₁-C₁₂-alkyl, aryl and their derivatives). R and R₁₋₆each can be the same or different each time they appear in the formula.perhalomethyl triazines

HX X = halogen, R = methyl, phenyl, C₁-C₁₂-alkyl, aryl and theirsubstituted derivatives. halobisphenyl A

HX o-nitrobenzaldehyde

sulfonates

RSO₃H R = CH₃, CF₃, Ph, C₁-C₁₂-alkyl, aryl and their substitutedderivatives imidylsulfonyl esters

RSO₃H

R = CH₃, CF₃, Ph, or C₁-C₁₂-alkyl, aryl and their substitutedderivatives. diaryliodonium salts

HX, BF₃ X = B(R₁)₄, Al(R₁)₄ (R₁ = halogen); R = H, halogen, NO₂, CN,SO₂R₅, OH, OCH₃, SCH₃, CF₃, OR₅, SR₅, CH₃, t-butyl, C₁-C₁₂- alkyl, aryland their substituted derivatives, NH₂, HNR₅, N(R₅)₂, (R₅ =C₁-C₁₂-alkyl, aryl and their substituted derivatives); COR₆ (R₆ = H,NH₂, HNR₅, OR₅, C₁-C₁₂-alkyl, aryl and their derivatives). R and R₁₋₆each can be the same or different each time they appear in the formula.sulfonium salts

HX, BF₃

X = B(R₁)₄, Al(R₁)₄ (R₁ = halogen); R = H, halogen, NO₂, CN, SO₂R₅, OH,OCH₃, SCH₃, CF₃, OR₅, SR₅, CH₃, t-butyl, C₁-C₁₂- alkyl, aryl and theirsubstituted derivatives, NH₂, HNR₅, N(R₅)₂, (R₅ = C₁-C₁₂-alkyl, aryl andtheir substituted derivatives); COR₆ (R₆ = H, NH₂, HNR₅, OR₅,C₁-C₁₂-alkyl, aryl and their derivatives). R and R₁₋₆ each can be thesame or different each time they appear in the formula. Y = O, S.diazosulfonate

RSO₃H R₁PhSO₃H R = phenyl, CH₃, CF₃, C₁-C₁₂-alkyl, aryl and theirsubstituted derivatives, R₁ = H, halogen, NO₂, CN, SO₂R₅, OH, OCH₃,SCH₃, CF₃, OR₅, SR₅, CH₃, t-butyl, C₁-C₁₂-alkyl, aryl and theirsubstituted derivatives, NH₂, HNR₅, N(R₅)₂, (R₅ = C₁-C₁₂-alkyl, aryl andtheir substituted derivatives); COR₆ (R₆ =H, NH₂, HNR₅, OR₅, C₁-C₁₂-alkyl, aryl and their derivatives). R and R₁₋₆ each can be the same ordifferent each time they appear in the formula. diarylsulfones

R = H, halogen, NO₂, CN, SO₂R₅, OH, OCH₃, SCH₃, CF₃, OR₅, SR₅, CH₃,t-butyl, C₁-C₁₂-alkyl, aryl and their substituted derivatives, NH₂,HNR₅, N(R₅)₂, (R₅ = C₁-C₁₂-alkyl, aryl and their substitutedderivatives); COR₆ (R₆ = H, NH₂, HNR₅, OR₅, C₁-C₁₂- alkyl, aryl andtheir derivatives). R and R₁₋₆ each can be the same or different eachtime they appear in the formula. 1,2-diazoketones

R, R₁ = H, halogen, NO₂, CN, SO₂R₅, OH, OCH₃, SCH₃, CF₃, OR₅, SR₅, CH₃,t-butyl, C₁-C₁₂-alkyl, aryl and their substituted derivatives, NH₂,HNR₅, N(R₅)₂, (R₅ = C₁-C₁₂-alkyl, aryl and their substitutedderivatives); COR₆ (R₆ = H, NH₂, HNR₅, OR₅, C₁-C₁₂- alkyl, aryl andtheir derivatives). R and R₁₋₆ each can be the same or different eachtime they appear in the formula. R, R₁ can be the same or different, orcan be connected through covalent bonds. R, R₁ = aryl, alkyl, and theirsubstituted derivatives. examples of diazoketones: 2- diazo-1-oxo-5-sulfonyl or 2-diazo- 1-oxo-4-sulfonyl naphthanol esters

R₁, R₂ = H, SO₂R (R = C₁-C₁₂-alkyl, aryl, and their substitutedderivatives).

examples of diazoketones: diazomethyl ketone

examples of diazoketones: diazo- Meldrums' acid

arylazide derivatives

RCO₂H or R = C₁-C₁₂-alkyl, aryl, and their substituted derivatives; R₁ =H, C₁- C₁₂-alkyl, aryl, and their substituted derivatives.

arylazide derivatives

HNR₂R₃ R = NR₂R₃, (R₂, R₃ = H, C₁-C₁₂-alkyl, aryl, and their substitutedderivatives), R₁ = H, C₁-C₁₂-alkyl, aryl, and their substitutedderivatives. benzocarbonates or carbamates

RCO₂H or HNR₁R₂ R = NR₁R₂ (R₁, R₂ = H, C₁-C₁₂-alkyl, aryl and theirsubstituted derivatives), C₁-C₁₂-alkyl, aryl and their substitutedderivatives. dimethoxybenzoinyl carbonates or carbamates

RCO₂H or HNR₃R₄ R = NR₃R₄ (R₃, R₄ = H, C₁-C₁₂-alkyl, aryl and theirsubstituted derivatives), C₁-C₁₂-alkyl, aryl and their substitutedderivatives; R₁, R₂ = H, C₁-C₁₂-alkyl, COPh, aryl and their substitutedderivatives. o-nitrobenzyloxycarbonates or carbamates

R₅CO₂H R₅PO₃H R₅SO₃H CF₃SO₃H or HNR₆R₇ R = COR₅ (R₅ = CF₃, OR₆, NH₂,HNR₆, C₁-C₁₂-alkyl, aryl and their derivatives (R₆ = H, C₁-C₃-alkyl,aryl and their substituted derivatives)), SO₂R₅, PO₂R₅, CONR₆R₇ (R₇ = H,C₁-C₃-alkyl, aryl and their substituted derivatives), R₁, R₂ = H,halogen, NO₂, CN, SO₂R₅, OH, OCH₃, OR_(a), N(R_(a))₂, (R_(a) =C₁-C₃-alkyl, aryl and their substituted derivatives); CH₃, t-butyl,C₁-C₁₂-alkyl, aryl and their substituted derivatives; R₃, R₄ = H,C₁-C₁₂-alkyl, aryl, and their substituted derivatives.nitrobenzensulphenyl

RCO₂H or HNR₁R₂ R = CF₃, NR₁R₂ (R₁, R₂ = H, C₁-C₃-alkyl, aryl and theirsubstituted derivatives), C₁-C₁₂-alkyl, aryl and their derivatives.o-nitroanilines

RCO₂H or HNR₄R₅ R = CF₃, NR₄R₅ (R₄, R₅ = H, C₁-C₁₂-alkyl, aryl and theirsubstituted derivatives), alkyl, aryl and their derivatives; R₁, R₂ = H,halogen, NO₂, CN, SO₂R₄, OH, OCH₃, OR_(a), N(R₄)₂; CH₃, t-butyl, C₁-C₁₂-alkyl, aryl and their substituted derivatives; R₃ = H, C₁-C₁₂-alkyl,aryl and their substituted derivatives.

TABLE 1B Examples of Radiation Sensitizers for PGR Reactions^(a)photosensitizer

Photosensitizers include but not limited to the following: benzophenone,acetophenone, benzoinyl C₁-C₁₂-alkyl ethers, benzoyl triphenylphosphineoxide, anthracene, thioxanthone, chlorothioxanthones, pyrene, Ru²⁺complexes, their various substituted derivatives, and the like.

TABLE 1C Examples of Stabilizers for PGR Reactions^(a) R—H stabilizer

R—H stabilizers include but not limited to the following: propylenecarbonate, propylene glycol ethers, t-butane, t-butanol, thiols,cyclohexene, their substituted derivatives and the like. ^(a)Substitutedderivatives contain at least one of the substituent groups, whichinclude but not limited to halogen, NO₂, CN, OH, SH, CF₃, C(O)H,C(O)CH₃, C₁-C₃-acyl, SO₂CH₃, C₁-C₃—SO₂R₂, OCH₃, SCH₃, C₁-C₃—OR₂,C₁-C₃—SR₂, NH₂, C₁-C₃—NHR₂, C₁-C₃—N(R₂)₂ (R₂ = # alkyl, can be the sameor different each time they appear in the formula).

In some embodiments of the present invention, PGR precursors are used incombination with co-reagents, such as radiation sensitizers. Onespecific example is the use of photosensitizers, which are compounds oflower excitation energies than the PGR used. Irradiation excitesphotosensitizers, which in turn initiate conversion of PGR precursors togive PGR. The effect of the photosensitizer is to shift the excitationwavelength used in photochemical reactions and to enhance the efficiencyof the formation of photo-generated reagents. Accordingly, in oneembodiment, the present invention makes use of, but is not limited to,photosensitizers as co-regents in PGR reactions. Many radiationsensitizers are known to those skilled in the art and include thosepreviously mentioned. It is to be understood that one of ordinary skillsin the art will be able to readily identify additional radiationsensitizers based upon the present disclosure.

In preferred embodiments of the present invention, the substrate surfaceis solid and substantially flat. As non-limiting examples, the substratecan be a type of silicate, such as glass, Pyrex or quartz, a type ofpolymeric material, such as polypropylene or polyethylene, and the like.The substrate surfaces are fabricated and derivatized for applicationsof the present invention.

Photo-generated acid (PGA) Deprotection and Oligonucleotide Synthesis

According to one embodiment of the present invention (FIGS. 1 and 2),linker molecules are attached to a substrate surface on whicholigonucleotide sequence arrays are to be synthesized (the linker is an“initiation moiety” a term also broadly including monomers or oligomerson which another monomer can be added). The methods for synthesis ofoligonucleotides are known, McBride et al., Tetrahedron Letter 24,245-248 (1983). Each linker molecule contains a reactive functionalgroup, such as 5′-OH, protected by an acid-labile protecting group 100.Next, a photo-acid precursor or a photo-acid precursor and itsphotosensitizer (Table 1) are applied to the substrate. A predeterminedlight pattern is then projected onto the substrate surface 110. Acidsare produced at the illuminated sites, causing cleavage of theacid-labile protecting group (such as DMT) from the 5′-OH, and theterminal OH groups are free to react with incoming monomers (FIG. 2,“monomers” as used hereafter are broadly defined as chemical entities,which, as define by chemical structures, may be monomers or oligomers ortheir derivatives). No acid is produced at the dark (i.e.non-illuminated sites) and, therefore, the acid labile protecting groupsof the linker molecules remain intact (a method of preventing H⁺diffusion between adjacent sites will be described later). The substratesurface is then washed and subsequently contacted with the first monomer(e.g., a nucleophosphoramidite, a nucleophosphonate or an analogcompound which is capable of chain growing), which adds only to thedeprotected linker molecules under conventional coupling reactionconditions 120. A chemical bond is thus formed between the OH groups ofthe linker molecules and an unprotected reactive site (e.g., phosphorus)of the monomers, for example, a phosphite linkage. After proper washing,oxidation and capping steps, the addition of the first residue iscomplete.

The attached nucleotide monomer also contains a reactive functionalterminal group protected by an acid-labile group. The substratecontaining the array of growing sequences is then supplied with a secondbatch of a photo-acid precursor and exposed to a second predeterminedlight pattern 130. The selected sequences are deprotected and thesubstrate is washed and subsequently supplied with the second monomer.Again, the second monomer propagates the nascent oligomer only at thesurface sites that have been exposed to light. The second residue addedto the growing sequences also contains a reactive functional terminalgroup protected by an acid-labile group 140. This chain propagationprocess is repeated until polymers of desired lengths and desiredchemical sequences are formed at all selected surface sites 150. For achip containing an oligonucleotide array of any designated sequencepattern, the maximum number of reaction steps is 4×n, where n is thechain length and 4 is a constant for natural nucleotides. Arrayscontaining modified sequences may require more than 4×n steps.

PGA Activated Coupling Reaction and Oligonucleotide Synthesis

According to another embodiment of the present invention (FIG. 3), aphoto-activator precursor, such as a compound containing tetrazolelinked to a photolabile group, is used. Linker molecules are attached toa substrate surface, on which oligonucleotide sequence arrays are to besynthesized 300. Acid labile protection groups on linkers aredeprotected 310. Next, a photo-activator precursor or a photo-activatorprecursor and its photosensitizer (Table 1) are applied to thesubstrate. A predetermined light pattern is then projected onto thesubstrate surface 320. At the illuminated sites, activator molecules areproduced and monomers are coupled to the linker. At the non-illuminatedsites, no activator molecules are produced and, therefore, no reactionoccurs (a method of preventing activator diffusion between adjacentsites will be described later). After proper washing, oxidation andcapping steps, the addition of the first residue is complete.

The attached nucleotide monomer also contains a protected functionalterminal group. The substrate containing the array of growing sequencesis then contacted with a second batch of acid 330. Sequences aredeprotected and the substrate is washed and subsequently contacted withthe second monomer. Again, the second monomer propagates only at thesurface sites that have been exposed to light 340. This chainpropagation process is repeated until polymers of desired lengths andchemical sequences are formed at all selected surface sites 350.

Alternative Embodiments of Oligonucleotide Synthesis UsingPhoto-generated Reagents

In some embodiments of the present invention, the appropriate monomersused in the coupling steps 120, 140, 150, 320, 340 and 350 arenucleotide analogs. The reaction of these monomers proceeds as describedin FIGS. 1 and 3 to give oligonucleotides containing modified residues.

In some embodiments of the present invention, the appropriate monomersused in the coupling steps 120, 140, 150, 320, 340 and 350 are thosecontaining an acid labile protecting group, such as DMT, at the 3′-OHposition. The reaction of these monomers proceeds as described in FIGS.1 and 3 but with sequence grown in an opposite orientation compared tothat using 5′-OH protected monomers. Such syntheses produceoligonucleotides containing a terminal 3′-OH, which are of particularuse as primers for in situ polymerase chain reactions (PCR).

Photo-generated Reagents and Photosensitizers

The use of PGR in the present invention permits chemical/biochemicalreactions under conventional conditions. The occurrence of the reactionis controlled, however, by in situ formation of at least one reagentupon irradiation. In some embodiments, irradiation is from a lightsource emitting UV and visible light. Heat, IR and X-ray irradiation arealso sources of irradiation. A PGR is produced by irradiation of a PGRprecursor or a photosensitizer (which in turn transfers its energy to aPGR precursor). Chemical transformation occurs to yield at least oneproduct (PGR), which is an intermediate or a stable compound. PGR isfrom part of the PGR precursor molecule dissociated from the parentstructure or a rearranged structure of the PGR precursor. PGR may be anacid, a base, a nucleophile, an electrophile, or other reagents ofspecific reactivities (Table 1).

In some embodiments of the present invention, improved reaction yieldsand/or suppression of side reactions are achieved by pre-irradiationactivation of at least one PGR before mixing with other reactants.Pre-irradiation activation allows time for active reactionintermediates, such as free radical species generated duringirradiation, to diminish and for products, such as H⁺, to reach a stableconcentration. Improved reaction yields and/or suppression of sidereactions are also achieved if at least one suitable stabilizer is used.One example is to provide at least one reagent to reduce the lifetime ofactive reaction intermediates such as a free radical species generatedduring irradiation, and to provide a low energy source of hydrogen. Thisis illustrated by the following reactions of generating H⁺ fromsulfonium salts (Ar₃S⁺X⁻).

RH compounds in the above equation are stable and are good H donors.Examples of such compounds include propylenecarbonate (one of the majorcomponents of UVI 6974 and UVI 6990), t-butane, cyclohexene, and thelike (Table 1C).

Photo-acid precursors within the scope of the present invention includeany compound that produces PGA upon irradiation. Examples of suchcompounds include diazoketones, triarylsulfonium, iodonium salts,o-nitrobenzyloxycarbonate compounds, triazine derivatives and the like.Representative examples of these compounds are illustrated in Table 1A.The table is compiled based on data found in following references: Süset al., Liebigs Ann. Chem. 556, 65-84 (1944); Hisashi Sugiyama et al.,U.S. Pat. No. 5,158,885 (1997); Cameron et al., J. Am. Chem. Soc. 113,4303-4313 (1991); Fréchet, Pure & Appl. Chem. 64, 1239-1248 (1992);Patchornik et al., J. Am. Chem. Soc. 92, 6333-6335 (1970).

An example of a photo-acid precursor is triarylsulfoniumhexafluoroantimonate derivatives (Dektar et al. J. Org. Chem. 53,1835-1837 (1988); Welsh et al., J. Org. Chem. 57, 4179-4184 (1992);DeVoe et al., Advances in Photochemistry 17, 313-355 (1992)). Thiscompound belongs to a family of onium salts, which undergophotodecompositions, either directly or sensitized, to form free radicalspecies and finally produce diarylsulfides and H⁺ (see above).

Another example of a photo-acid precursor is diazonaphthoquionesulfonatetriester ester, which produces indenecarboxylic acid upon UV irradiationat λ>350 nm. The formation of the acid is due to a Wolff rearrangementthrough a carbene species to form a ketene intermediate and thesubsequent hydration of ketene (Süs et al., Liebigs Ann. Chem. 556,65-84 (1944); Hisashi Sugiyama et al., U.S. Pat. No. 5,158,885 (1997)).

These photolytic intermediates and products have been extensively usedin cationic and radical catalyzed polymerizations for high-resolutionmicroimaging photolithograpy.

Photo-acid precursor compounds have been widely used for many years inprinting and microelectronics industries as a component in photoresistformulations (Willson, in “Introduction to microlithography”, Thompsonet al. Eds., Am. Chem. Soc.: Washington D. C., (1994)). These reactionsare, in general, fast (complete in a matter of seconds or minutes),proceed under mild conditions (room temperature, neutral solution), andthe solvents used in the photoreactions (haloalkanes, ketones, esters,ethers, toluene, and other protic or aprotic polar solvents) arecompatible with oligonucleotide (McBride et al., Tetrahedron Letter 24,245-248 (1983))⁶ or other organic solution chemistry. Among thephoto-generated acids listed in Table 1, selections are made forchemistry compatibility to minimize side reactions. The chemicalproperties, such as acidity of the photo-generated acids can be adjustedby different substitution groups on the ring or chain moieties. Forinstance, the electronegative sulfonate group in the indenecarboxylicacid formed helps to stabilize the negative charge on the carboxylicgroup attached to the same ring moiety to give an acid that effectivelydeprotects the 5′-O-DMT group (FIG. 2) in a way comparable to that ofusing the conventional trichloroacetic acid (TCA). In general,electron-withdrawing groups, such as O₂SOR, NO₂, halogens, C(═O)R(R=aryl, alkyl, and their substituted derivatives, or XR₁ (X=S, O, N;R₁=aryl, alkyl, and their substituted derivatives) increase the strengthof the corresponding acids. Electron donating groups, such as OR(R=aryl, alkyl, and their substituted derivatives), decrease thestrength of the corresponding acids. The availability of acids ofdifferent strengths provides a repertoire of reagents for a range ofacid-catalyzed deprotection reactions.

Photo-base precursors within the scope of the present invention includeany compound that produces PGB upon irradiation. Examples of suchcompounds include o-benzocarbamates, benzoinylcarbamates,nitrobenzyloxyamine derivatives listed in Table 1, and the like. Ingeneral, compounds containing amino groups protected by photolabilegroups can release amines in quantitative yields. The photoproducts ofthese reactions, i.e., in situ generated amine compounds, are, in thisinvention, the basic reagents useful for further reactions.

Photo-reagent precursors within the scope of the present inversioninclude any compound that produces a reagent required by achemical/biochemical reaction upon irradiation. Examples of suchcompounds include 1-(dimethoxylbenzoinyl)tetrazole (heterocycliccompound tetrazole is a PGR), dimethoxylbenzoinyl OR₁ (R₁OH is a PGR,R₁=alkyl, aryl and their substituted derivatives), sulfonium salts(thiol ether Ar₂S is a PGR), and the like.

Photosensitizers within the scope of the present invention include anycompound that are sensitive to irradiation and able to improveexcitation profile of PGR by shifting its excitation wavelength andenhancing efficiency of irradiation. Examples of such compounds includebenzophenone, anthracene, thioxanthone, their derivatives (Table 1B),and the like.

Alternative Applications of PGR

In one embodiment of the present invention, photo-generated reagents(Table 1) are applied to on-chip parallel synthesis of peptide arraysusing amino acid monomers containing reactive functional groupsprotected by t-Boc (acid labile) or Fmoc (base labile) groups (FIG. 4).The methods of peptide synthesis are known, Sterwart and Young, “Solidphase peptide synthesis”, Pierce Chemical Co.; Rockford, Ill. (1984);Merrifield, Science 232, 341-347 (1986); Pirrung et al., U.S. Pat. No.5,143,854 (1992). According to one embodiment of the present invention,linker molecules are attached to a substrate surface on which peptidesequence arrays are to be synthesized. Each linker molecule contains areactive functional group, such as an —NH₂ group, protected by the acidlabile t-Boc group 500. Next, a photo-acid precursor or a photo-acidprecursor and its photosensitizer are applied to the substrate. Apredetermined light pattern is then projected onto the substrate surface505. At the illuminated sites, acids are produced, the acid labileprotecting groups, such as t-Boc, are cleaved from the N-terminal NH₂thereby enabling it to react with incoming monomers (FIG. 4). At thedark sites, no acid is produced and, therefore, the acid labileprotecting groups of the linker molecules remain intact. The substratesurface is then washed and subsequently supplied with the first monomer(a protected amino acid, its analogs, or oligomers), which adds only tothe deprotected linker molecules under conventional coupling reactionconditions 510. A chemical bond is thus formed between the NH₂ group ofthe linker molecules and the carbonyl carbon of monomers to afford anamide linkage. After proper washing steps, the addition of the firstresidue is complete. The attached amino acid monomer also contains areactive functional group protected by the acid labile t-Boc group. Thesubstrate containing the arrays of the growing sequences is thensupplied with a second batch of a photo-acid precursor and exposed to asecond predetermined light pattern 515. The selected sequences aredeprotected and the substrate is washed, and supplied, subsequently,with the second monomer. Again, the second monomer propagates only atthe surface sites that have been exposed to light. The second residueadded to the growing chain also contains a reactive functional groupprotected by an acid-labile group 520. This chain propagation process isrepeated until polymers of desired lengths and chemical sequences areformed at all selected surface sites 525. For a chip containing apeptide array of any designated sequence pattern, the maximum number ofreaction steps is 20×n, where n is the chain length and 20 is aconstant, the number of naturally occurring amino acids. Arrayscontaining modified amino acids may require more than 20×n steps.

In another preferred embodiment of the present invention (FIG. 6), aphoto-base precursor, such as an amine protected by a photo-labilegroup, is applied to solid surface loaded with linkers 600. Each linkermolecule contains a reactive functional group, such as NH₂, protected bya base-labile group. Next, a photo-base precursor, such as(((2-nitrobenzyl)oxy)carbonyl)-piperidine (Cameron and Fréchet, J. Am.Chem. Soc. 113, 4303-4313 (1991))⁸, is applied to the substrate. Apredetermined light pattern is then projected onto the substrate surface605. At the illuminated sites, bases are produced, causing cleavage ofthe base-labile protecting groups from the linker molecules, and theterminal NH₂ groups are free to react with incoming monomers. At thedark sites, no base is produced and, therefore, the base labileprotecting groups of the linker molecules remain intact. The substratesurface is then washed and subsequently supplied with the first monomercontaining a carboxylic acid group, which adds only to the deprotectedlinker molecules under conventional coupling reaction conditions toafford an amide linkage 610. After proper washing, the addition of thefirst residue is completed. The attached amino acid monomer alsocontains a reactive functional terminal group protected by a base-labilegroup. The substrate containing the arrays of the growing sequences isthen supplied with a second batch of a photo-base precursor and exposedto a second predetermined light pattern 615. The selected sequences aredeprotected and the substrate is washed, and subsequently supplied withthe second monomer. Again, the second monomer propagates only at thesurface sites that have been exposed to light. The second residue addedto the growing sequences also contains a reactive functional terminalgroup protected by a base-labile group 620. This chain propagationprocess is repeated until polymers of desired lengths and desiredchemical sequences are formed at all selected surface sites 625.

The present invention is not limited to the parallel synthesis of arraysof oligonucleotides and peptides. The method is of general use in solidphase synthesis of molecular arrays where complex synthesis patterns arerequired at each step of chain extension synthesis. One specific exampleis synthesis of oligosacchride arrays containing sequences of diversecarbohydrate units and branched chains (FIG. 7). According to thepresent invention, a photo-acid precursor is applied to a solid surfacecontaining protected carbohydrates. Each carbohydrate molecule containsseveral reactive OH groups, each of which is protected by protectinggroups. Each of these protecting groups requires different deprotectionconditions. A predetermined light pattern is then projected onto thesubstrate surface. At the illuminated sites, acid is produced and theprotection groups labile under a particular set of conditions arecleaved. Deprotected OH groups are free to react with an incomingmolecule. At the dark sites, no acid is produced and, therefore, theacid labile protecting groups of the carbohydrate molecules remainintact. The substrate surface is then washed and subsequently suppliedwith a monomer (a carbohydrate or oligosacchride), which adds only tothe deprotected OH under conventional reaction conditions to afford aglycosidic linkage. Wong et al., J. Am. Chem. Soc. 120, 7137-7138(1998). These steps are repeated to give oligosacchrides containingvarious glycosidic linkages at the first deprotected OH position. Next,a photo-base precursor is applied to the substrate. A secondpredetermined light pattern is then projected for the second time ontothe substrate surface. At the illuminated sites, base is produced andthe protection groups labile under this condition are cleaved.Deprotected OH groups of the second batch are free to react with anincoming molecule. At the dark sites, no base is produced and,therefore, the base labile protecting groups of the carbohydratemolecules remain intact. The substrate surface is then washed andsubsequently supplied with a second monomer, which adds only to thesecond deprotected OH of the second time under conventional reactionconditions to afford a glycosidic linkage. These steps are repeated togive oligosacchrides containing various glycosidic linkages at thesecond deprotected OH position. Branched oligosacchrides are formed. Incontinued synthesis, various PGR are used to achieve selectivedeprotection of the OH protecting groups until desired oligosacchridearrays are synthesized.

The present invention enables use of photo-generated reagents in morecases than just deprotection reactions to achieve selective reaction inaccordance with a predetermined pattern without changing the course ofwell-developed conventional chemistry. Furthermore, the presentinvention is not limited to deprotection reactions, photo-generatedreactive compounds, such as alcohols (ROH, R=alkyl, aryl and theirsubstituted derivatives), can be used as reagents for a variety ofchemical conversions, such as esterification, nucleophilic substitutionand elimination reactions. These reactions are important steps forfabrication of custom MMA-chips.

Synthesis Apparatus

FIGS. 8A thought 8C illustrate three embodiments of the programmable,light-directed synthesis apparatus of this invention. As shown in FIG.8A, the apparatus is comprised of four sections: a reagent manifold 812,an optical system, a reactor assembly, and a computer 814.

Reagent Manifold

The reagent manifold 812 of FIG. 8A performs standard reagent metering,delivery, circulation, and disposal. It consists of reagent containers,solenoid or pneumatic valves, metering valves, tubing, and processcontrollers (not shown in FIG. 8A). The reagent manifold 812 alsoincludes an inert gas handling system for solvent/solution transport andline purge. The design and construction of such a manifold are wellknown to those who are skilled in the art of fluid and/or gas handling.In many cases, commercial DNA/RNA, peptide, and other types ofsynthesizers can be used as the reagent manifold 812 of this invention.

Optical System

The function of the optical system shown in FIG. 8A is to producepatterned light beams or light patterns 807 c for initiatingphotochemical reactions at predetermined locations on a substratesurface 810 a. The optical system shown in FIG. 8A is comprised of alight source 802, one or more filters 803, one or more condenser lenses804, a reflector 805, a Digital Micromirror Device (DMD) 801, and aprojection lens 806. During operation, a light beam 807 a is generatedby the light source 802, passes through the filter(s) 803, and becomes alight beam 807 b of desired wavelength. A condenser lens 804 and areflector 805 are used to direct the light beam 807 b on to the DMD 801.Through a projection lens 806, DMD projects a light pattern 807 c on thesubstrate surface 810 a of a reactor 810. Details about the DMD 801 aredescribed below.

A light source 802 may be selected from a wide range of light-emittingdevices, such as a mercury lamp, a xenon lamp, a halogen lamp, a laser,a light emitting diode, or any other appropriate light emitter. Thewavelengths of the light source 802 should cover or fall within theexcitation wavelengths of the concerned photochemical reaction. Thepreferred wavelengths for most of the concerned photochemical reactionsare between 280 nm and 500 nm. The power of the light source 802 shouldbe sufficient to generate a light pattern 807 c intense enough tocomplete the concerned photochemical reactions in a reactor 810 within areasonable time period. For most applications, the preferred lightintensity at the substrate surface 810 a position is between 0.1 to 100mW/cm². For many applications, a mercury lamp is preferred due to itsbroad wavelengths and availability of various powers.

Selection criterions for a filter(s) 803 are based on the excitationwavelength of concerned photochemical reactions and otherconsiderations. For example, it is often desirable to remove undesirablyshort and long wavelengths from the light beam 807 a in order to avoidunwanted photo-degradation reactions and heating in a reactor 810. Forexample, in the synthesis of oligonucleotides and other bio-relatedmolecules, it is preferred to remove wavelengths shorter than 340 nm. Toavoid heating, an infrared cut-off filter is preferably used to removewavelengths beyond 700 nm. Therefore, more than one filter may beneeded.

A key component in the Optical System shown in FIG. 8A is a DigitalMicromirror Device 801, which is used to generate light patterns 807 b.A DMD is an electronically controlled display device and it is capableof producing graphical and text images in the same manner as a computermonitor. The device is commercially available from Texas InstrumentsInc., Dallas, Tex. USA, for projection display applications (Hornbeck,L. J., “Digital light processing and MEMS, reflecting the digitaldisplay needs of the networked society,” SPIE Europe Proceedings, 2783,135-145 (1996)). Each DMD 801 contains a plurality of small andindividually controllable rocking-mirrors 801 a, which steer light beamsto produce images or light patterns 807 c.

DMD 801 is a preferred means of producing light patterns in the presentinvention for several reasons. First, it is capable of handlingrelatively short wavelengths that are needed for initiating concernedphotochemical reactions. Second, the device has high optical efficiency.Third, it can produce light patterns of high contrast ratio. Inaddition, devices of high resolution formats (up to 1920×1080) have beendemonstrated. These features permit one to conveniently generate opticalpatterns for the synthesis of practically any desired molecular sequencearray by using the photochemistry described in this invention. In thisaspect, the apparatus of this invention is highly flexible as comparedwith the prior art method of producing sequence arrays using photomasks.

Other types of electronically controlled display devices may be used forgenerating light patterns. FIG. 8B illustrates an exemplary embodimentof the present invention, using a reflective liquid crystal arraydisplay (LCD) device 821. Reflective LCD devices are commerciallyavailable from a number of companies, such Displaytech, Inc. Longmont,Colo. USA. Each reflective LCD device 821 contains a plurality of smallreflectors (not shown) with a liquid crystal shutter 821 a placed infront of each reflector to produce images or light patterns.High-resolution devices, up to 1280×1024, are already available fromDisplaytech. The optical system shown in FIG. 8B is like that of thedevice of FIG. 8A except for the optical arrangement for directing lightonto display devices. A beam splitter 825 is used in the optical systemshown in FIG. 8B to effectively couple light onto and out of flatreflects.

In another embodiment of the present invention, a transmissive LCDdisplay 841 is used to generate light patterns, as shown in FIG. 8C. Atransmissive LCD display 841 contains a plurality of liquid crystallight valves 841 a, shown as short bars in FIG. 8C. When a liquidcrystal light valve 841 a is on, light passes; when a liquid crystallight valve is off, light is blocked. Therefore, a transmissive LCDdisplay can be used in the same way as an ordinary photomask is used ina standard photolithography process (L. F. Thompson et al.,“Introduction to Microlithography”, American Chemical Society,Washington, D.C. (1994)). In FIG. 8C, a reflector 845 is used to directa light beam 847 b to the transmissive LCD display 841.

Most commercially available display devices, including DMD, reflectiveLCD, and transmissive LCD are designed for handling visible light (400nm to 700 nm.) Therefore, when these commercially available displaydevices are used, the best operation mode of the programmable,light-directed synthesis apparatus of this invention is achieved whenthe excitation wavelength of the photo-reagent precursors is between 400nm and 700 nm. However, the use of the instrument and the methods ofthis invention extends beyond the above wavelength range.

FIGS. 8A through 8C depict apparatus designs for making one array chipat a time. The present invention also encompasses devices for producinga plurality of chips. FIG. 12 schematically illustrates amechanical/optical stepping mechanism for enhancing the throughput andthe efficiency of the synthesis apparatus of this invention. In thisstepping mechanism, a light beam 1204 a is projected from a displaydevice, (not shown in the figure,) passes through a projection lens1202, and is directed by a reflector 1203 towards a reactor 1201 aforming an image or a light pattern 1204 b. The reflector 1203 has arotating mechanism that can direct the light pattern 1204 b towards anyone of the several surrounding reactors 1201 a through 1201 f. In aregular synthesis process of, for example, oligonucleotides, the lightpattern 1204 b is directed towards a specific reactor, e.g. 1201 a, onlyduring a photochemical deprotection reaction step. Then the lightpattern 1204 b is directed towards other reactors, while reactor 1201 agoes through the rest of synthesis steps, such as flushing, coupling,capping, etc.

Other stepping mechanisms may also be used in the present invention. Forexample, a step-and-repeat exposure scheme, which is routinely used inphotolithography of semiconductors, may be used. General descriptions ofstep-and-repeat photolithography were given by L. F. Thompson et al., inIntroduction to Microlithography, American Chemical Society, Washington,D.C. (1994). In this scheme, a large substrate containing multiplereaction-well arrays is used. The substrate is mounted on a x-ytranslation stage. At each step, an optical exposure covers one orseveral arrays. Then, the substrate is moved to the next position andanother optical exposure is performed. The process is repeated until allreaction-well arrays are exposed.

The present invention is not limited to the use of electronicallycontrolled display devices as the means of generating photolithographypatterns. Conventional photomasks, which are made of glass plates coatedwith patterned chromium or any other appropriate films, may be used aswell. In this case, the transmissive LCD display device 841 shown inFIG. 8C is replaced with a conventional photomask while rest of theapparatus remains the same. The use of conventional photomasks ispreferred for the production of a large number of the same products. Aconventional photomask may contain a large number of array patterns sothat a large number of molecular arrays can be synthesized in parallel.However, for small batch production of various different array productsthe use of electronically controlled display devices is much preferreddue to its flexibility.

Reactor Configuration

As described in earlier sections, photogenerated reagents involved inthe current invention are in solution phase. When the reagents are usedto produce spatially defined patterns, such as arrays, appropriatemeasures should be taken to spatially isolate individual elements. FIGS.9A through 9C schematically illustrate three preferred embodiments ofisolation mechanisms of the present invention. In the embodiment shownin FIG. 9A, a transparent substrate 901 and a cap 902 form a reactioncell or a reactor, which is filled with a solution containing one ormore photo-reagent precursors. Reactionwells, bounded by barriers 903,are embossed on the cap 902. The cap 902 is preferably made of a plasticor an elastomer material inert to all chemicals involved in thereaction. Before a photolytic reaction takes place, the cap 902 ispushed against the substrate 901 forming contacts between the barriers903 and the substrate and isolates of individual reaction-wells. Lightbeams are then projected into a number of selected reaction-wells 904 aand 904 c, as shown in Step 3 of FIG. 9A. Photolytic and otherphoto-reagent-induced reactions take place in the light-exposedreaction-wells 904 a and 904 c while there is no photo activate reactionin the unexposed reaction-well 904 b. When properly constructed andoperated, the isolation mechanism described prevents diffusion ofreagents across individual reaction-wells. In addition, the spacebetween adjacent reaction-wells 904 b and 904 c provides a buffer zone904 d to further prevent any inter-mixing between reaction-wells.

The buffer zone 904 d, shown in FIG. 9A, provides space for additionmechanisms of preventing interference among individual reaction-wells.FIG. 10 illustrates detailed structure of the reaction-wells of thecurrent invention in a three-dimensional perspective view. The figureshows that the buffer zones (labeled as 1006 in FIG. 10) are allinterconnected. This interconnected structure permits one to flush thebuffer zone with appropriate solutions while all the reaction-wells areclosed. In a preferred method, buffer zone 904 d is flushed after thecompletion of the photolytic and photo-reagent-induced reactions andbefore the lifting of the cap 902, with a solution that would eitherquench the photo-reagent-induced chemical reactions or neutralize thephotogenerated reagents inside the exposed reaction-wells 904 a and 904c. The spillover of the photogenerated reagents from the exposedreaction-wells 904 a and 904 c would thus not cause undesirable chemicalreactions in other areas after the cap 902 is lifted. For neutralizing aphotogenerated acid, a weak basic solution, such as pyridine in CH₂Cl₂,may be applied. For quenching nucleotide-coupling reaction, acetonitrileor other suitable solvents may be used.

FIG. 9B illustrates another embodiment of the isolation mechanism of thepresent invention. In this embodiment, reaction-well structures, orreaction-well barriers 913, are constructed on a transparent substrate911 while the cap 912 has a flat inner surface. The substrate 911 ispreferably made of glass. The cap 912 is preferably made of a plastic oran elastomer material inert to all chemicals involved in the reactions.The seal mechanism and the preferred operation mode are similar to thosedescribed earlier for the embodiment shown in FIG. 9A.

FIG. 9C illustrates the third embodiment of the isolation mechanism ofthe present invention. In this embodiment, a pattern of non-wetting film933 is coated on the surface of a transparent substrate 931. During anoperation, a reactor is first filled with a solution 934. Then thesolution 934 is drained from the reactor and droplets are formed on thesubstrate 931 surface because the solution wets the substrate 931surface but not the non-wetting film 933 surface. The droplets areisolated from each other. Light beams 935 can then be projected ontopredetermined droplets 934 a and 934 c to initiate photolytic and otherphoto-reagent-induced reactions. This embodiment eliminates the need fora sealing mechanism and is suitable for large-scale biochip productionusing large substrates. The use of non-wetting films to confine fluid iswell-know in the art and has been described by Thomas M. Brennan in U.S.Pat. No. 5,474,596 for the synthesis of DNA oligomers using aninkjet-printing method.

Reactors of this invention are preferably assembled into a cartridgeform as illustrated in FIG. 10. The design shown in the figure utilitiesthe isolation mechanism shown in FIG. 9B. Other isolation mechanisms,such as the ones shown in FIGS. 9A and 9C, can be easily implementedinto similar cartridge forms. As shown in FIG. 10, each cartridgecontains a transparent substrate 1001, which can be made of glass orpolymer materials of suitable chemical and optical properties. Above thesubstrate is a barrier layer 1003 containing pluralities of openings toform arrays of isolated reaction-wells 1004. In principle, thereaction-wells can be of any reasonable shapes and sizes. Circular andsquare wells are preferred. Most preferably, wells are of circular shapeof 10 to 1,000 μm in diameter and 5 to 100 μm in depth. For example, ina specific design, circular reaction-wells are 140 μm in diameter, 20 μmin depth, and are arranged as an orthogonal array with equalcenter-to-center distance of 200-μm. With this design, 2,500reaction-wells are packed into an area of one square centimeter. In eachreaction well, about 6.4 fmol molecules may be synthesized, assuming theaverage distance between immobilized adjacent molecules is 20 Å. Thevolume of the reaction-well is about 300 pico-liter, providingsufficient volume required for reactions. The barrier layer 1003 is madeof opaque materials, such as metals or blackened polymers, to opticallyisolate individual reaction-wells from each other. The third layer is areactor cap 1002. The cap 1002 has three functions: reactor enclosure,reagent connection/distribution, and reaction-well isolation. The cap1002 is preferably made of a polymer material that is flexible andresistant to chemicals/solvents involved in the concerned synthesisprocesses. The material may be selected from a group of polymersincluding polyethylene, polypropylene, polyethylene-polypropylenecopolymer, fluorinated polymers and various other suitable ones. Thereagent inlet 1012 and outlet 1013 are placed at two opposite ends ofthe reactor. Branching channels 1011 are made to distribute reagentsevenly across the reactor. The center region of the cap is a pad 1015that can be pushed down to tightly seal the reaction-wells 1004 below.Immediately above the reactor there is a mechanical actuator (not shownin FIG. 10 but shown in FIGS. 8A through 8C as 811, 831, and 851), whichcan be, for example, driven either solenoidally or pneumatically. Theactuator can either push the pad of the reactor cap to seal allreaction-wells or retract to open all the reaction-wells. This operationis to accommodate the sealing mechanism shown in FIGS. 9A and 9B. Theinset in FIG. 10 shows an enlarged view of the reaction-well structures,which contain extruded rims 1005 to facilitate sealing. While not shownin FIG. 10, the reactor substrate contains alignment marks, which permitthe alignment of the reactor in an optical lithography system of thepresent invention.

The reactor cartridge shown in FIG. 10 is most suitable for use in anordinary chemical and biochemical laboratory environment. The enclosedconstruction of the cartridge prevents chemical and particulatecontamination from the environment. In order to achieve the best andconsistent results, the cartridges are preferably manufactured in acontrolled environment to ensure the chemical integrity inside thecartridge. The cartridges are then filled with an inert gas, such as Ar,and sealed by plugging the inlet and outlet of the reactor. Then thecartridges can be stored and/or shipped to user laboratories.

Reactor Fabrication

The reactors of the present invention (FIGS. 9A through 9C and 10) canbe fabricated using various well-known microfabrication processes, suchas photolithography, thin film deposition, electroplating, and molding(M. Madou, Fundamentals of Microfabrication, CRC Press, New York,(1997)). These techniques have been widely used for making various ofmicrofluidics devices, electromechanical devices, chemical sensors, andoptical micro-devices. For example, the reaction-well structure shown inFIG. 10 can be fabricated by using electroplating of suitable metalfilms on a glass substrate. At the end of this description, an exampleis given to demonstrate the fabrications processes involved. Thereaction-well structures on a glass substrate may also be made usingchemical etching processes, which have been widely used to make variousmicrofluidics devices (Peter C. Simpson et al. Proc. Natl. Acad. Sci.,95: 2256-2261 (1998)).

Reactor cap 1002, shown in FIG. 10, can be fabricated using a precisionmolding process. Such a process is widely available in plasticfabrication industry. The polymer material used is preferably in blackcolor to minimize light reflection and scattering during light exposure.Welding and adhesive bounding methods can be used to assemble theplastic cap 1002 and a substrate 1001 into an integrated cartridge.

Making non-wetting film patterns on glass and other substrates is awell-known art in many fields (Uthara Srinivasan et al., Proc. IEEESolid-State Sensors and Actuators, June 1991, 1399-1402). The film isusually formed by a monolayer of self-assembled molecules (SAM) or athin polymer film of low surface energy material such as Teflon. Themost frequently used SAMs on glass substrates include varioushydrocarbon alkylsilanes and fluoroalkylsilanes, such asoctadecyltrichlorosiliane and 1H, 1H, 2H, 2H-perfluorodecyltrichlorosiliane. The patterning process involves the useof photoresists and photolithography. Example VII at the end of thisdescription provides a detailed patterning procedure. Thin polymerfilms, such as Teflon, can be printed onto glass and plastic surfaces byusing a screen printing process. The screen printing process is awell-know art in printing industry and in electronic industry. Generalprocedures of screen printing for microfabrication applications aredescribed by M. Madou in Fundamentals of Microfabrication, CRC Press,New York, (1997). In addition, hydrophobic printed slides arecommercially available from vendors, such as Erie Scientific Company,Portsmouth, N.H. USA. When non-wetting film patterned substrates areused, the reactor configuration can be simplified because thereaction-well-sealing mechanisms shown in FIGS. 8A through 8C and FIG.10 are no longer needed.

Control of the Apparatus

As illustrated in FIG. 8A, the synthesis apparatus of the presentinvention is controlled by a computer 814, which coordinates the actionsof the DMD 801, the seal actuator 811 of the reactor 810, and thereagent manifold 812. In case of synthesizing oligonucleotides, duringmost of synthesis steps, the synthesis apparatus operates as aconventional synthesizer and the computer 814 controls reagent manifold812 to deliver various reagents to the reactor 810. At a photo-directeddeprotection step, the reagent manifold 812 delivers a photo-acidprecursor into the reactor 810. The computer 814 activates the sealactuator 811 to isolate reaction-wells and, then, sends data to DMD 801to project a light pattern 807 c onto the reactor 801. At the completionof the photoreactions, the light pattern 807 c is switched off, aquenching solution is delivered into the reactor 801, the seal actuator811 is lifted, and the synthesis control system resumes the steps ofconventional synthesis.

Variations and Modifications

Many variations and applications of the present invention are possible.FIG. 11A illustrates a variation of reaction-well structure. A masklayer 1103 is added to the bottom of the reaction well. One or moreopenings, which occupy a total one tenth to one half of thereaction-well surface area, are made on the masks for light 1104 to passthrough. The mask layer 1103 is preferably made of a thin and chemicalresistant metal film, such as Cr. On top of the metal film, a SiO₂ film(not shown) is deposited to facilitate immobilization of linkermolecules. This reaction-well design permits the spatial separation of aphotochemical reaction and photogenerated-reagent-induced chemicalreactions. FIG. 11A illustrates a photo-acid induced chemical reaction.Upon a light exposure, protons H⁺ are produced from a photo-acidprecursor in the open areas. The protons, then, diffuse into surroundingareas in the well to cleave acid-labile protecting groups P_(a) onimmobilized oligomer molecules 1106. This arrangement helps to minimizethe contact between photo-generated radical intermediates and theoligomers and thus, to suppress undesirable side-reactions that mightoccur due to the presence of radical intermediates.

FIG. 11B illustrates another variation of the reaction-well structureand light exposure strategy. This embodiment is also designed todecrease the possibility of undesirable side-reactions due radicalintermediates. Only a fraction of the reaction-well surface is exposedto light 1114. The chance for undesirable side-reactions in other areasis, consequently, decreased.

The applications of the chemical processes and the apparatus (FIG. 8Athrough 8C) of the present invention extend beyond the fabrication ofmolecular arrays. For example, the apparatus using DMD 801 shown in FIG.8A may be used as a general-purpose assay apparatus for studyingchemical and biochemical reactions. The Digital Micromirror Device 801controls precisely and simultaneously light dosages in all individualreaction-wells of a reactor 810. This feature allows one to preciselycontrol the production of photogenerated reagent in all reaction-wellsand, therefore, to perform a large-scale, parallel assay.

Obviously many modifications and variations of this invention arepossible in the light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

The invention is further described by the following Examples, which areprovided for illustrative purposes only and are not intended nor shouldthey be construed as limiting the invention in any manner. Those skilledin the art will appreciate that variations on the following Examples canbe made without deviating from the spirit or scope of the invention.

EXAMPLE I Photo-acid Generation

This experiment demonstrates efficient generation of H⁺ upon lightirradiation of a PGA as monitored by increased values of the chemicalshift of the H₂O signal as a function of light irradiation time.

Six samples containing a sulfonium salt (0.4% of 50% triaryl sulfoniumhexaflurophosphate in propylene carbonate, Secant Chemicals, Boston,Mass.) in 0.5 mL CD₂Cl₂ were placed in nuclear magnetic resonance (NMR)tubes. A reference one-dimensional (ID) spectrum of these samples wasrecorded (600 MHz NMR spectrometer, Bruker, Karlsruhe, Germany) usingmethod well known to those skilled in the art. One of the samples wasthen irradiated using a collimated light source (22 mW, Oriel, Stanford,Calif.) at 365 nm for a defined length of time (FIG. 13A) and 1D NMRspectrum was recorded immediately after the irradiation. A second samplewas then irradiated at 365 nm for a second defined length of time (FIG.13A) and 1D NMR spectrum was recorded immediately after the irradiation.These experiments were repeated for each of the samples prepared. Foreach NMR spectrum, chemical shift of the H₂O was measured. In theabsence of light, H₂O signal appeared at 1.53 ppm. Upon irradiation,this signal moved to a higher ppm value (down field shifted) due to thegeneration of H⁺.

In FIG. 13B the correlation of the changes in chemical shift of the H₂Osignal with irradiation time is plotted. The formation of H⁺ under theconditions used follows a first order kinetics relationship and theapparent rate constant for formation of H⁺ derived is 1.3×10^(−2±0.06)s⁻¹.

EXAMPLE II Deprotection of Nucleostide Monomers Using PGA

These experiments demonstrate efficient deprotection of the DMT group on5′-OH of nucleosides using PGA.

Two samples were prepared in which DMT-G attached to (controlled porousglass, 0.2 μmol (CPG) added to sulfonium salt (0.4% of 50% triarylsulfonium hexaflurophosphate in propylene carbonate, Secant Chemicals,Boston, Mass.) in 0.5 mL CH₂Cl₂. One sample was irradiated using a UVlamp (UVGL-25, 0.72 mW) at 365 nm for 2 min, while the other sample, acontrol, was of irradiated Upon completion of the irradiation, CPG waswashed with CH₂Cl₂ and CH₃CN, followed by treatment with concentratedaqueous NH₄OH (1 mL) for 2 h at 55° C. The solution was brieflyevaporated in vacuo. A buffer solution (0.1 M triethylammonium acetate(TEAA), 15% in CH₃CN) was added to the CPG sample and the resultantsolution was injected into a C18 reverse phase (10 μm, μ-bondapak,Waters) HPLC column. A gradient of 0.1M TEAA in CH₃CN was used to elutethe sample. Authentic samples of DMT-dG and dG were used as referenceand co-injection of PGA deprotected dG and authenic dG confirms theresult of the PGA reaction. FIGS. 14A1 and 14A2 show HPLC profiles ofDMT-dG and the PGA deprotected dG.

The same procedures were performed for DMT-dC, DMT-dG, DMT-dA, andDMT-rU. FIGS. 14B1 and 14B2 show HPLC profiles of DMT-rU and the PGAdeprotected rU.

Other photo-acid precursors, such as 2,1,4-diazonaphthoquionesulfonatetriester, triaryl sulfonium hexafluroantimonate and hexaflurophosphate(Secant Chemicals, Boston, Mass.), and perhalogenated triazine (MidoriKagaku), were also used for these deprotection reactions. Completedeprotection of the DMT group was achieved with these photoacidprecursors.

EXAMPLE III Deprotection of Nucleoside Monomers Using Pre-activated PGA

This experiment demonstrates that pre-activation of PGA precursor is aneffective means of reducing side reactions in deprotection using PGA.Depurination due to cleavage of glycosidic bonds in nucleotides underacidic conditions is a known problem. This problem is exacerbated in theuse of PGA for deprotection since at the initiation of reaction, theamount of H⁺ requires time to build up. The following experiment is toshow that this problem can be alleviated using a pre-activated PGA.

The samples and experimental conditions used in this experiment were asdescribed in Example II, except that the PGA solution (0.4% of 50%triaryl sulfonium hexafluroantimonate in propylene carbonate) was firstirradiated at 365 nm for 2 min. before adding the CPG attachedDMT-nucleoside.

Pre-irradiation (UVGL-25, 0.72 mW) at 365 nm for 2 min was perform usinga PGA solution (0.4% of 50% triaryl sulfonium hexafluroantimonate inpropylene carbonate). The irradiated solution was then added to powderDMT-dA (approximately 1 μmol). The solution was incubated for anadditional 2 min. 1D NMR spectrum was recorded using methods well knownto those skilled in the art. Another sample of DMT-dA (1 μmol) was mixedwith a PGA solution (0.4% of 50% triaryl sulfonium hexafluroantimonatein propylene carbonate) and the mixture was irradiated (UVGL-25, 0.72mW) at 365 nm for 2 min. 1D NMR spectrum was recorded. Depurinationcauses gradual disappearance of the signals of dA. The comparison of thetwo NMR spectra recorded for these experiments indicates less sidereactions for the reaction using pre-activated PGA.

EXAMPLE IV Oligonucleotide Synthesis Using PGA

These experiments demonstrate efficient synthesis of oligonucleotides onCPG support using PGA. Oligonucleotides of various sequences (A, C, G,and T) and chain lengths (n=2-8) were synthesized using photo-acidprecursors on a Perspective synthesizer (Perspective Biosystems,Framingham, Mass.).

Synthesis of DMT-TTTT (FIG. 15A2), was carried out on a 0.2 μmol scaleaccording to the protocol in Table 2. This is a direct adoption of theconventional phosphoramidite synthesis but with minor modifications atstep 2. At this step, a PGA (0.4% of 50% triaryl sulfoniumhexaflurophosphate in propylene carbonate) was added and the reactioncolumn was irradiated with 365 nm light for 2 min. The column wasextensively 515 wash with solvents after the photo-deprotectionreaction. Upon completion of the synthesis, the sequence was cleavedfrom CPG and deprotected using concentrated NH₄OH. The sample wasexamined using C18 reverse phase HPLC using a TEAA in CH₃CN gradient.The HPLC profile of the crude product of DMT-TTTT synthesized using aPGA is shown (FIG. 15A1). FIG. 15A2 shows DMT-TTTT using theconventional trichloroacetic acid (TCA) deprotection chemistry. FIGS.15B1 and 15B2 show HPLC profiles of the crude octanucleotides which wassynthesized using the PGA approach.

TABLE 2 Protocol of Automated Oligonucleotide Synthesis (0.2 μmol)¹Amount Vol. Time Conc. Used (ml) (sec) (mM) (μmol) A. Using PGA  1detritylation² 1% UVI-6974/ 1.20 180 100  114 CH₂Cl₂ (v/v)  2a wash³CH₃CN 2.40 200  2c wash CH₂Cl₂ 2.00 50  3 coupling A. tetrazole/ 0.10 2450  45 CH₃CN  4 coupling A. tetrazole/ 0.10 2 450  45 CH₃CN  5(simultan- B. monomer/ 0.10 2 100  10 eous) CH₃CN  6 coupling B.tetrazole/ 0.10 63 450  45 CH₃CN  7 wash CH₃CN 0.04 31  8 wash CH₃CN0.66 17  9 capping A. acetic 0.15 4  10% 147 anhydride/ lutidine/THF 10(simultan- B. N-methylimi- 0.15 4  10% 183 eous) dazole/THF 11 washCH₃CN 0.10 15 12 wash CH₃CN 0.27 7 13 oxidation l₂/THF/pyridine/ 0.29 7 0.02 6 H₂O 14 wash CH₃CN 0.29 7 15 capping A. acetic 0.13 3 127anhydride/ lutidine/THF 16 (simultan- B. N-methylimi- 0.13 3 158 eous)dazole/THF 17 wash CH₃CN 0.57 15 total (sec) 612 total (min) 10.2 B.Using Conventional TCA  1 detritylation 3% TCA 1.20 59 100  114  2 washCH₃CN 1.00 20  3 coupling A. tetrazole/ 0.10 2 450  45 CH₃CN  4 couplingA. tetrazole/ 0.10 2 450  45 CH₃CN  5 (simultan- B. monomer/ 0.10 2 100 10 eous) CH₃CN  6 coupling B. tetrazole/ 0.10 63 450  45 CH₃CN  7 washCH₃CN 0.04 31  8 wash CH₃CN 0.66 17  9 capping A. acetic 0.15 4  10% 147anhydride/ lutidine/THF 10 (simultan- B. N-methylimi- 0.15 4  10% 183eous) dazole/THF 11 wash CH₃CN 0.10 15 12 wash CH₃CN 0.27 7 13 oxidationl₂/THF/ 0.29 7  0.02 6 pyridine/H₂O 14 wash CH₃CN 0.29 7 15 capping A.acetic 0.13 3 127 anhydride/ lutidine/THF 16 (simultan- B. N-methylimi-0.13 3 158 eous) dazole/THF 17 wash CH₃CN 0.57 15 total (sec) 261 total(min) 4.35 ¹Protocol is adopted from an Expedite 8909 synthesizer usedfor oligonucleotide synthesis using PGA deprotection. ²Highlighted stepsfor incorporation of the PGA reactions. Patterned light irradiation isapplied at this step. ³Washing step is being optimized at this time toreduce the cycle time.

EXAMPLE V Amino Acid Deprotection and Peptide Synthesis Using PGA

These experiments demonstrate efficient deprotection of the aminoprotection group using PGA in peptide synthesis.

A sample of 10 mg (10 μmol) of HMBA resin (Nova Biochem, La Jolla,Calif.) containing t-Boc-Tyr was employed. Deprotection was performed ina CH₂Cl₂ solution containing a PGA (10% of 50% triaryl sulfoniumhexafluroantimonate in propylene carbonate) by irradiating the samesolution at 365 nm for 15 min. The reaction was incubated for anadditional 15 min and the resin was washed with CH₂Cl₂. The possiblepresence of residual amino groups was detected using ninhydrin colortests and the result was negative. The resin was then washed and theamino acid cleaved from the resin using NaOH (0.1 M in CH₃OH). FIG. 16Bshows the HPLC profile of the PGA deprotected Tyr. FIG. 16A shows theHPLC profile of Tyr obtained using conventional trifluoroacetic acid(TFA) deprotection.

Synthesis of a pentapeptide, Leu-Phe-Gly-Gly-Tyr (SEQ ID NO:1), wasaccomplished using 100 mg of Merrifield resin. The PGA deprotection ofthe t-Boc group was performed and the resin was tested using ninhydrinuntil no color resulted. The coupling reaction was carried out usingconditions well known to those skilled in the art. The PGA deprotectionand coupling steps were repeated until the pentamer synthesis wascompleted. The sequence was cleaved from the resin and its HPLC comparedwell to that of the same sequence synthesized using conventional peptidechemistry.

EXAMPLE VI Fabrication of Microwells

Formation of microwells using the fabrication methods of the presentinvention is demonstrated in this example. FIG. 17A schematicallyillustrates the fabrication procedure used. In the first fabricationstep, a thin bimetal film 1702 of Cr/Cu 200/1000 Å thick was evaporatedon a glass substrate 1701 in a sputtering evaporator. he bimetal film1702 Cr provides good adhesion to the glass surface and Cu provides agood base for subsequent electroplating. The surface was thenspin-coated with a positive photoresist 1703 of 18 μm thick. Thephotoresist film 1703 was then patterned using photolithography(exposure to UV light using a photomask aligner and development).Electroplating using a plating solution for bright Ni was utilized toapply a plate a Ni film of 18 μm thick onto the exposed Cu surfaceresulting in microwell barriers 1704. The solution formula and platingconditions are as following. NiSO₄.6H₂O: 300 g/l, NiCl₂.6H₂O: 30-40 g/lBoric Acid: 40 g/l, Saccharin: 2-5 g/l, Butynediol (2-Butyne-1,4-diol):100 mg/l, Sodium lauryl sulfate: 50 ppm, pH: 3.0-4.2, Current density:10 A/dm², Temperature: 50° C. The photoresist film 1703 was thenstripped. Cu film was etched using a HNO₃:H₃PO₄:CH₃COOH=0.5:50.0:49.5(volume) solution and Cr film was etched using a HCl:H₃PO₄:CH₃COOH5:45:50 (volume) solution activated by an aluminum stick. A spin-onglass film was then coated on to the sample surface to form a SiO₂ film1705. FIG. 17B shows a photograph of the resulted microwell sample.

EXAMPLE VII Solution Isolation Using Patterned Non-wetting Films

This example illustrates that arrays of organic-solvent droplets wereformed on a glass surface patterned with non-wetting films using themethods taught in the present invention. FIG. 18A schematicallyillustrates a fabrication procedure for coating a patterned non-wettingfilm on a glass substrate 1801. A glass substrate 1801 was thoroughlycleaned in a warm H₂SO₄:H₂O=1:1 (volume) solution. The substrate 1801was then spin-coated with a positive photoresist of about 2.7 μm thick.The photoresist film 1802 was exposed to UV light using a photomaskaligner and developed. In this example, a photomask containing a matrixof circular dots were used and, therefore, the same pattern was formedin the photoresist film 1802. The patterned glass substrate was dippedinto a 1 mM FDTS (1H, 1H, 2H, 2H -perfluorodecyltrichlorosiliane)anhydrous iso-octane solution in a dry box and soaked for at least 10minutes. Then, the substrate was rinsed with iso-octane 2-3 timesfollowed by a thorough water rinse. The photoresist was stripped and aFDTS film was left on the glass surface as a non-wetting film.

Tests of wetting effects were performed in an enclosed cell to avoidevaporation of volatile solvents. During a test, the cell was filledwith a testing solvent or solution and then drained. Tests were made onvarious organic/inorganic solvents and solutions including CH₂Cl₂,CH₃CN, CH₃OH, CH₃CH₂OH, TCA/CH₂Cl₂ solution,I₂/tetrahydrofuran-water-pyridine solution, and other solutions involvedin oligonucleotide synthesis. Formation of droplet arrays was observedfor each testing solvent/solution. FIG. 18B shows a photograph ofmethanol droplet array formed on a non-wetting film patterned glassplate.

EXAMPLE VIII Array Synthesis on a Patterned Glass Substrate Using PGA

These experiments demonstrate the use of the method and instrument ofthe present invention in making molecular microarray chips.

Fabricated glass substrates containing isolated reaction wells atspecified areas as described in Example VI were employed. The glassplates were derivatized with linker molecules (10%N-(3-triethoxysilylpropyl)-4-hydroxylbutyramide in ethanol) containingfree OH groups. Synthesis on the glass substrate was performed using areactor and a digital light projector as described in this specificationand a DNA synthesizer (Perspective). Oligonucleotide synthesis wasaccomplished according to the protocol shown in Table 2. The glasssurface was first contacted with DMT-T phosphoramidite to couple thefirst residue. The sequences were treated, in subsequent steps, withcapping and oxidation reagents and washed with CH₃CN before and aftereach step of the reactions. The glass plate was then treated with a PGA(0.4% of 50% triaryl sulfonium hexaflurophosphate in CH₂Cl₂) deliveredby the synthesizer and exposed to computer generated patterned lightirradiation (30 s) from a collimated light source at 365 nm and 3 mw oflight source intensity, (Stanford, Calif.). The surface was thenextensively washed with CH₃CN. In the light exposed areas, free hydroxylgroups were generated. After oxidation and wash steps, the surface wascontacted by fluorescein-labeled phosphoramidite monomers in a secondcoupling step. The molecular arrays synthesized were treated with NaOHaqueous solution (0.1 M). The array contains fluorescence labeled dimerswere visualized under a fluoromicroscope (Bio-Rad, Richmond, Calif.).The results of which are shown in FIG. 19.

11 1 5 PRT Artificial Sequence Synthetic 1 Leu Phe Gly Gly Tyr 1 5 2 4DNA Artificial Sequence Synthetic 2 cgat 4 3 4 DNA Artificial SequenceSynthetic 3 tcac 4 4 4 DNA Artificial Sequence Synthetic 4 gact 4 5 4DNA Artificial Sequence Synthetic 5 atcc 4 6 4 PRT Artificial SequenceSynthetic 6 Val Asp Ala Glu 1 7 4 PRT Artificial Sequence Synthetic 7Thr Tyr Ala Gln 1 8 4 PRT Artificial Sequence Synthetic 8 Ile Tyr SerGlu 1 9 4 PRT Artificial Sequence Synthetic 9 Ala Asp Ser Gln 1 10 8 DNAArtificial Sequence Synthetic 10 tttttttt 8 11 8 DNA Artificial SequenceSynthetic 11 acgtacgt 8

We claim:
 1. A method of attaching monomers at specific reaction siteson a substrate, said specific reaction sites containing one or morenon-photolabile protected initiating moieties, the method comprising: a)contacting said substrate with a liquid solution comprising one or morephoto-reagent precursors, said precursors selected from the groupconsisting of acid and base precursors, such that said liquid solutionis in contact with said initiating moieties; b) isolating said specificreaction sites; c) irradiating a selected number of the isolatedreaction sites to produce, in situ, at least one photo-generated reagentwithout the formation of a polymeric coating layer, thereby directlydeprotecting the initiating moieties at the irradiated reaction sites soas to create deprotected initiating moieties; and d) contacting saidsubstrate with a first monomer, said first monomer comprising anunprotected reactive site and a protected reactive site, underconditions such that said unprotected reactive site of said monomercouples with said deprotected initiating moieties so as to create anattached first monomer.
 2. The method of claim 1, wherein saidinitiating moieties of step (a) comprise linker molecules, each of saidlinker molecules comprising a reactive functional group protected by anacid-labile protecting group.
 3. The method of claim 2, wherein saidreactive functional group of said linker molecules comprises a hydroxylgroup.
 4. The method of claim 1, wherein said first monomer is selectedfrom the group consisting of nucleophosphoramidites, nucleophosphonatesand analogs thereof.
 5. The method of claim 1, wherein said attachedfirst monomer of step (d) comprises a nucleotide monomer with aprotected group.
 6. The method of claim 5, wherein said protected groupis protected by an acid-labile group.
 7. The method of claim 5, furthercomprising the step of deprotecting said protected group so as to createa deprotected attached nucleotide monomer.
 8. The method of claim 7,further comprising the step of contacting said deprotected attachednucleotide monomer with a second monomer, said second monomer comprisingan unprotected reactive site and a protected reactive group, underconditions such that said unprotected reactive site of said secondmonomer couples with said deprotected attached nucleotide monomer so asto create an attached second monomer, said attached first and secondmonomers comprises a multimer.
 9. The method of claim 8, wherein saidmultimer comprises DNA.
 10. The method of claim 1, wherein saidattachment of said monomers occurs with a yield of 98% or greater. 11.The method of claim 1, wherein said initiating moieties of step (a)comprise oligomers to which a monomer can be attached.